Social relevance:

For 30 years, people have been confused about how much plain water to drink. Over 30 years, health professionals have criticized media advice to drink 8 glasses of water per day, citing lack of evidence (Valtin et al, 2002; Yamada et al, 2022). Health authorities have not set drinking water-specific recommendations, assuming 1) that any or all types of drinks hydrate equivalently, i.e. that people do not need to drink plain water to replace lost body water, and 2) the average healthy person can rely on thirst as guide for water intake.

The lack of drinking water-specific recommendations significantly impacts daily lives because it translates into limited or no support for drinking water in public health services, laws, and retail options.

Scientific relevance:

Thirst is considered the primary driver of water intake and main defense against body water deficit in healthy young adults (IOM, 2005). Health authorities set total water intake recommendations for the average healthy man and woman (e.g. 2.5 L/d for men and 2.0 L/d for women in Europe) but, additionally, advise people to use thirst as a guide for water intake, recognizing that individual water requirements vary widely (EFSA, 2010; IOM, 2005).

Although thirst can be satiated by water intake, it can also be ignored per custom (Greenleaf, 1992) or suppressed by an upward-shifted thirst threshold. The thirst threshold, the set-point where osmoreceptor cells shrink and release their neural or hormonal signal, is a function of the solute concentration or osmolality inside and outside the osmoreceptor cells (Nose et al, 1988a,b). Cells with higher intracellular solute content require a higher external osmolality to shrink.

Specific Aims The ultimate goal of this study is to address gaps in the literature about drinking water and check assumptions that limit the development of drinking water-specific recommendations. The study will examine if osmoadaptation to chronic hypertonicity, due to daily intake of hypertonic fluid sources, can explain suppressed thirst in healthy individuals under conditions of daily life. To facilitate causal inference about drinking water effects for long-term health, this study was designed to link experimental data about osmoadaptation at the cellular level with clinical data relevant for conditions of daily life in Salzburg Austria with population-based data about water intake and chronic disease risk in Salzburg Austria.

This study will test effects of drinking enough plain water to dilute urine everyday for 4 weeks (about 500 mL 4 times per day in summer). The study will include healthy, normal weight, young, men and women, who all usually meet European adequate intake recommendations for total water intake (TWI), but usually consume less than 1L/d PWI, and have biomarkers of chronic hypertonic stress (concentrated urine and saliva) for 4 consecutive weeks before starting the randomized study.

Osmoreceptor cells in the brain and periphery, which are collectively responsible for perceived thirst, accumulate intracellular solute (Bourque, 2008), such as amino acids, to adapt to chronic hypertonic stress. Increased protein breakdown is a well-established strategy for coping with chronic hypertonic stress, observed across species with drought/aestivation (Yancey et al, 1982). Evidence of increased protein breakdown is observed in patients with excess body water loss due to skin or renal damage (Kovarik et al, 2021) and with less than 2L/d usual total water intake in healthy young men (Stookey et al, 2023). Higher concentrations of intracellular solute allow cells to tolerate chronic hypertonic stress by creating an osmotic gradient that favors retaining water inside the cells.

Patients with chronic hypertonicity due to uncontrolled diabetes are known to develop suppressed thirst. Healthy athletes (Casa et al, 2000) and individuals exposed to heat (Rosinger et al, 2022) are known to experience 'involuntary dehydration' or incomplete rehydration after dehydration, when given ad-libitum fluids and allowed to drink following thirst. Thirst is significantly reduced in older adults (Phillips et al, 1984). It is plausible that decreased thirst is a function of intracellular osmolyte accumulation, resulting from altered metabolism in response to chronic hypertonicity.

In older adults, reduced thirst is attributed to higher baseline extracellular osmolality and higher osmotic set-point for thirst sensation (Kenney & Chiu, 2001). In older people, reduced thirst or faster thirst satiation after drinking water is related to a larger drop in activation of the anterior midcingulate cortex (aMCC) in the brain (Farrell et al, 2008).

With respect to young adults, while evidence indicates that 'involuntary dehydration' depends on cations lost or excreted from the intracellular and/or extracellular space (Nose et al, 1988), roles for osmolytes other than cations remain to be explored. There are gaps in the clinical literature regarding effects of chronic extracellular hypertonicity on osmoadaptation, shifted osmoreceptor set-point, suppressed thirst, and hydration biomarkers.

Chronic extracellular hypertonicity and suppressed thirst are conceivable in daily life, because people frequently consume foods and fluids that are more concentrated than blood (beverage osmolality >280 mmol/kg). Most commercially available beverages including milk and juice have an osmolality above 300 mmol/kg.

Given that adaptation to chronic hypertonicity carries metabolic cost (Pena-Villalobos et al, 2016) and favors chronic disease risk factors in healthy young adults (Stookey et al, 2023), including micronutrient (e.g. Zn) excretion (Zorbas et al, 1993; Zorbas et al, 1995), oxidative stress, protein breakdown, and altered immune function, low thirst in young adults may not be a reliable guide for water intake - if thirst is 'suppressed' as opposed to 'satiated'. On the contrary, low thirst in young adults may signal chronic suboptimal cell hydration and unmet need for hypotonic water.

Hypotheses

Holding constant usual intake of food and other beverages and physical activity levels over 10 weeks, this study hypothesizes that participants who are randomly assigned to drink water to dilute afternoon urine to USPG<1.013 daily (PWI of about 20 mL/kg or 500 mL 3x/d in Spring and Autumn; 4x/d in Summer) for 4 weeks will have a:

Primary outcome

• significantly greater increase in the mean overnight water restricted thirst rating between Week 5 and Week 10 compared to participants assigned to the control group.

Secondary outcomes

• significantly greater decrease between Week 5 and Week 10 in the acute decrease in regional cerebral blood flow seen by functional MRI in brain regions of interest (S1/M1, prefrontal cortex, anterior midcingulate cortex, premotor cortex, and superior temporal gyrus) from maximum thirst after overnight water restriction to immediately following 500 mL drinking water, compared to the control group.
• significantly different metabolomic profile in Week 10, with greater shift away from the aestivation- and Warburg-like patterns, including significantly greater reduction in protein breakdown between Week 5 and Week 10, compared to the control group.
• significantly greater decrease in urine excretion of zinc between Week 5 and Week 10, compared to the control group.

All randomized participants must have complete data from 4 weeks of screening. All inclusion criteria below must be met:

• 4 weeks of 3-day mean total water intake of 35 ml/kg body weight
• 4 weeks of <1L/d drinking water
• 4 weeks of <1 hr/d moderate or vigorous physical activity given daily life conditions in Salzburg, Austria
• No history of chronic disease
• Normal blood pressure,
• Normal blood chemistry and complete blood count (CBC) test results
• Normal overnight urine concentration, and normal acute urine dilution after 500mL drinking water
• Normal weight and height between 160-170 cm for women and 175-185 cm for men)
• After overnight food and water restriction urine osmolality above 800 mmol/kg, salivary osmolality above 100 mmol/kg, and a thirst score below 70 mm on a 100 mm visual analog scale.

Exclusion Criteria:

• Individuals will be excluded from participation if the gender-specific target numbers have already been reached

• Perceived stress score of > 20 at any time during Weeks 1-4

  • Schedule that does not allow continuous study participation
  • Intention to spend more than a day outside the Salzburg area during the study period (to reduce exposure to ground water with different background D2O as potential source of error)

• Headache within the last six months

• Pregnant, planning a pregnancy, or irregular or unknown menstrual cycle

  • Active use of tobacco or e-cigarettes
  • Daily consumption of alcohol
  • Regular use of medication
  • Agoraphobia or claustrophobia that would prevent brain fMRI testing
  • Metal-containing intrauterine device (IUD) that would prevent fMRI testing
  • Doubtful or unwilling to complete all study procedures (including drinking alcohol; drinking plain tap water, blood tests, 24-hour urine collection, fMRI scans, and stable isotope tests)
  • Unwilling to be randomly assigned to intervention or control
  • Incomplete baseline data in Weeks 1-4 (including blood tests, 24-hour urine collection, and MRI scans).

Only de-identified data will be shared with research partners.

Individuals processing diet and activity data and lab samples.